Quantitative Proteomic Analysis of RHDV2-Infected Rabbit Liver

RHDV2-infected and uninfected control rabbit liver samples [described in a previous study (Neave et al., 2018 (link))] were used for label-free quantification (LFQ). Briefly, rabbits were either infected with RHDV2 (genotype GI.1bP-GI.2) or mock-infected with PBS. The animals were humanely killed 24 h post-infection, and 25% liver homogenates were prepared in RNA-later buffer and stored at −20°C. Samples from three RHDV2-infected animals (K375, K376, and K378) and three uninfected animals (K3, K14, and K12) were used in this study. Liver samples were clarified at 0.2 g for 5 min at 4°C, and 100 mg of solid precipitate was collected into tubes with glass beads. Liver homogenates were lysed in 300 μl of PBS (pH 7.5) with 150 mM NaCl, 0.5 mM EDTA, 0.5% Igepal CA-630 with protease inhibitors (Roche, Basel, Switzerland), 1 mM MgCl₂, and 1 μl/ml benzonase nuclease (5 KU; Sigma-Aldrich). The suspension was homogenized using a Precellys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and incubated on ice for 30 min with gentle rocking to allow for full lysis. Finally, the lysates were cleared by centrifugation and supernatants were used for MS sample preparation.